Sequencing device

ABSTRACT

A method of preparing reagents includes inserting a cartridge into an instrument. The cartridge includes a plurality of reagent enclosures disposed in a cavity of the cartridge and exposing a port to an exterior of the cartridge. Each reagent enclosure includes a reagent container including a reagent and an internal cavity defining a compressible volume, an opening defined through the reagent container to the internal cavity. The method further includes connecting a plurality of fluid ports to the openings of the plurality of reagent enclosures; applying a solution through the fluid ports to at least partially fill the plurality of reagent enclosures; and cycling a pressure of the cavity, whereby for each of the reagent enclosures, during increasing pressure, the solution enters the internal cavity of the reagent container, combines with the reagent, and compresses the compressible volume, and during decreasing pressure, the compressible volume decreases and the reagent is ejected through the opening.

CROSS-REFERENCE TO RELATED APPLICATION(S)

This application is a divisional application under 35 U.S.C. § 120 ofU.S. application Ser. No. 14/742,404 filed Jun. 17, 2015 and now U.S.Pat. No. 9,890,424, which application claims benefit under 35 U.S.C. §119(e) of U.S. Provisional Application No. 62/013,475 filed Jun. 17,2014. The entire contents of the aforementioned applications areincorporated by reference herein.

FIELD OF THE DISCLOSURE

This disclosure, in general, relates to improved sequencing devices.

BACKGROUND

Increasingly, biological and medical research is turning to sequencingfor enhancing biological studies and medicine. For example, biologistand zoologist are turning to sequencing to study the migration ofanimals, the evolution of species, and the origins of traits. Themedical community is turned sequencing for studying the origins ofdisease, sensitivity to medicines, and the origins of infection. But,sequencing has historically been an expensive process, thus limiting itspractice.

BRIEF DESCRIPTION OF THE DRAWINGS

The present disclosure may be better understood, and its numerousfeatures and advantages made apparent to those skilled in the art byreferencing the accompanying drawings.

FIG. 1 includes an illustration of an example sequencing instrument.

FIG. 2 includes a schematic of an example sequencing instrument.

FIG. 3 includes a schematic of an example sequencing instrument.

FIG. 4 is a perspective view describing an exemplary reagent storageapparatus.

FIG. 5 is a perspective view describing an example container.

FIG. 6 is a cross-sectional perspective view describing an examplecontainer.

FIG. 7 is an exploded schematic view describing an example container.

FIG. 8 is a detailed perspective view describing an example container.

FIG. 9 is a perspective view describing an exemplary reagent storageapparatus.

FIG. 10 is a perspective view describing an example container.

FIG. 11 is a cross-sectional perspective view describing an examplecontainer.

FIG. 12, FIG. 13, FIG. 14, FIG. 15, and FIG. 16 include illustrations ofan exemplary cartridge for enclosing one or more enclosures.

FIG. 17 and FIG. 18 illustrate a schematic of an example method.

FIG. 19 is an exploded schematic view describing an example valve.

FIG. 20 is a cross-sectional schematic view describing an example valve.

FIG. 21 is a cross-sectional schematic view describing an example valve.

FIG. 22 is a cross-sectional schematic view describing an example valve.

FIG. 23 and FIG. 24 include illustrations of an example manifold andcartridge.

FIG. 25 includes an illustration of an example fluidic circuit.

FIG. 26 includes a flow diagram of an example method for preparing areagent solution.

FIG. 27 includes a flow diagram of an example method for measuring ananalyte.

FIG. 28 includes a flow diagram of an example method for preparing areagent cartridge.

The use of the same reference symbols in different drawings indicatessimilar or identical items.

DETAILED DESCRIPTION

In an exemplary embodiment, a sequencing system includes an instrumentto receive a semiconductor sequencing chip and to carry out processesthat result in the identification of a sequence of bases. In particular,the instrument can receive a reagent cartridge, wash solutions and asemiconductor sequencing chip. The instrument can include a userinterface, such as a touchscreen user interface, and can includecomputational circuitry and controllers to control the delivery ofreagents and wash solution to the semiconductor sequencing chip, as wellas the acquisition of data from the semiconductor sequencing chip tofacilitate identification of the sequence of bases.

An exemplary instrument includes a reagent cartridge receptacle, afurther receptacle for receiving a wash buffer cartridge, and a chipclamp for receiving a semiconductor sequencing chip. In addition, theinstrument includes a touchscreen user interface. Such an instrumentprovides for limited sequencer touch points, no compressed gasutilization (instead utilizing a closed pump-driven system), nohigh-quality water utilization, easy operation (including an intuitivegraphical user interface, plug-and-play consumables, etc.), fast singleday runtimes, integrated onboard computing for primary data analysis,dual-mode operation in a research use only (RUO) or diagnostic mode(Dx), small benchtop footprint, scalability (upgradable chassis fordifferent performance levels) or a low-cost, or any combination thereof.

In particular, the instrument can include a compressor for providinginternally generated gas pressure. Reagents can be provided in apreloaded cartridge to limit user interaction with reagent preparation.Similarly, wash solutions can be provided as a plug-and-play washsolution, such as within a cartridge. In an example, the pH of the washsolution can be stabilized using a solid-state buffer.

Flow rates of reagents within the system can be controlled for exampleusing dynamic flow control using a pinch valve regulator. The system canalso include a cleaning solution that performs automatic post-runwashing. The instrument may include an internal server for processingdata received from the semiconductor sequencing chip. Alternatively, orin addition, the system can provide an output data port to connect anexternal server for processing the data. Furthermore, the internalcomputational systems can be configurable and upgradable.

The system can utilize pressure driven liquid flow using an internal gassupply, without the use of external gas supplies. In particular, areagent cartridge system can utilize liquid or lyophilized nucleotideswithin separate enclosures or bags. The enclosure can be deflated of anyinitial air content by pressurizing the chamber external to the bags andflowing air from the bags to waste. The wash solution can be appliedinto the bags. Residual air bubbles can rise to the top and can purge towaste. Mixing is accomplished by pressurizing and rapidly depressurizingthe chamber external to the bags, causing the liquid in the bags topressurizing and depressurized. The liquid or lyophilized nucleotidesare contained within the mixer (reagent container) that in response todepressurization expels the solution including nucleotides, causing amixing of the nucleotide within the bag.

As external gas pressure is applied to the bag enclosure, internal washsolution flows into the mixer and an internal compressible volume withinthe mixer compresses. When the gas pressure external to the bag isquickly released, the pressure charged within the compressible volumeforces the wash solution out through nozzles at high velocity, mixingthe liquid including nucleotides within the bag.

When applying flow through a flow cell of the semiconductor sequencingchip, dynamic flow control can be accomplished by utilizing a pinch flowregulator (pinch valve regulator). Such a dynamic flow control reducesthe use of resistance tubing coils and reduces the potential forclogging. Flow rates can be programmable and can be adjusted byadjusting a control pressure within the pinch flow regulator.

The system can utilize a solid-state buffering system, such as a ceramicbuffering system, for example, particulate titanium dioxide. The washsolution reagent can be provided in a single use bottle that is readilymixed. The solution can include a solid-state buffer, eliminating autopH routines, and providing long-term pH stability. The particulatetitanium dioxide can be easily confined to the bottle by a filter, thuslimiting the particulate within the system. In addition, the primarywash bottle can utilize packaging that has low permeability to gas andthus is low permeability to acidifying CO₂. The container may also beshipped with a CO₂ absorbent packet, to further limit exposure to carbondioxide.

The semiconductor sequencing chip can be received in a chip clamp thatloads the chip in the system and connects the chip to the fluidicssystem. The chip clamp can include an integrated squid valve manifoldand removes tubing connections. The chip clamp can further includereference electrodes, integrated chip temperature control, andintegrated manifold heating.

The instrument can further rely on a reagent cartridge, limiting contactand processing by users. The simple cartridge is loaded and includesfive containers or enclosures (for nucleotide and a bead find orpH-adjusting reagent). The cartridge can also include a charging port toquickly pressurize/depressurize the cartridge utilizing an exhaustvalve, for example on a manifold connecting to the reagent cartridge.Optionally, a silencer is applied on the exhaust to reduce noiseassociated with a quick depressurizing the cartridge.

The instrument further includes electronics that are scalable andconfigurable. In particular, computing power and memory can beexchanged. The system can further include support for RFID tags, forexample, on semiconductor sequencing chips or cartridges.

An exemplary reagent cartridge can include color-coded ports fordifferent nucleotide solutions and a bead find or pH adjusting solution.In addition, the reagent cartridge can include input and output portsfor a CO₂ scrubber. The cartridge can also be provided with an RFID tagthat identifies, for example, lot numbers and expiration dates.

The cartridge can include individualized chambers into which reagentcontainers are applied or the CO₂ scrubber is inserted. The reagentcontainers are inserted through the cartridge lid into reagent pouchesor enclosures secured to the lid within the cartridge.

For exemplary reagent cartridge manufacturing, fittings are applied intopouches, for example, including a low-density polyethylene/poly ethyleneterephthalate film. The cartridge base, lid, port gaskets, and otherparts are then assembled applying the pouches to the lid, followed bythe lid to the base, as well as the insertion of gaskets into the lid. Areagent container includes a mixer body into which a foam member isinserted. A mixer cap is applied to the mixer body. In an example, themixer cap can include lyophilized reagent or liquid reagent. In anotherexample, the reagent can be frozen or stored within porous ceramic orpolymeric foam. The assembled reagent mixers and scrubbers can then beapplied to the reagent cartridge assembly. RFID tags can be applied tothe cartridge, and the cartridge can be boxed and stored for shipping.

Reagent can be applied in to the mixer or cartridges either in alyophilized form or as a frozen liquid form. In an example, lyophilizednucleotide pellets can be formed and then inserted into the mixers. Inanother example nucleotides can be dried onto filter paper that isintegrated into bug mixers. In another example, nucleotides can be drieddirectly onto compressible foam or within the caps (second portions) ofthe mixer (reagent containers).

In an example, FIG. 1 includes an illustration of exemplary instrument100 for sequencing. The instrument 100 can include a connector toreceive a container of buffered solution 104, a clamp 106 to receive asequencing device, and a manifold to receive a reagent cartridge 102.Further, the sequencing instrument 100 includes computational circuitryto control fluid flow, data retrieval from the sequencing device,interpretation of the data, and a user interface 108. In a particularexample, the sequencing device is a pH or ion sensitive device, forexample, including a plurality of ion sensitive field effect transistors(ISFETs).

In a particular example, the sequencing instrument includes circuitry tocontrol fluid flow within the system. In an example illustrated in FIG.2, the instrument 200 includes connections to a cartridge 202. Reagentcan flow from the cartridge 202 through a fluidic circuit 204 and to asequencing device 206. Fluid passing through the fluidic circuit 204 canoptionally be directed to a waste container 212 or through thesequencing device 206 to a waste container 210 through pinch flowregulators 208 or 230. In an alternative example, a single wastecontainer can take the place of waste containers 210 or 212. Anexemplary embodiment of a cartridge 202 is illustrated in FIGS. 12-16.An exemplary embodiment of a pinch flow regulator is illustrated FIGS.19-22. An exemplary fluidic circuit is illustrated in FIG. 3 and FIG.25.

As further described below, a solution in a container 226, such as abuffered solution, can flow through a valve 228 and be used to preparereagent solutions in reagent enclosures 214. The reagent solutions 214can selectively flow to the fluidic circuit 204 and to the sequencingdevice 206 or waste containers 210 or 212. The solution in the solutioncontainer 226 can optionally flow through valve 232 to the fluidiccircuit 204 and can act as a wash solution, washing the fluidic circuit204 and optionally the sequencing device 206 of reagents from thereagent solutions. The buffered solution in the solution container 226can optionally be pumped in the system. Alternatively, the bufferedsolution can be driven by pressure, for example, supplied using airthrough inlet 234.

In an example, the system 200 can include a compressor 216 thatcompresses gas or air to flow through a scrubber cartridge 220,optionally included in the reagent cartridge 202. For example, thescrubber cartridge 220 can include components, such as soda lime, toremove carbon dioxide from air. Receptacle 218 can be used to store andsupply pressurized air to the system. For example, the pressurized aircan be used to pressurize the solution container 226. In anotherexample, the pressurized air can be supplied to the cavity of thecartridge 202 to pressurize the reagent enclosures 214 and drive reagentsolutions selectively through the valves 222 to the fluidic circuit 204.In a further example, the compressed air from the receptacle 218 can beused to clean a fluidic circuit 204 through valve 224. The pressurizedair can drive remaining liquids within the fluidic circuit 204 to thewaste container 212, through the sequencing device 206 to the wastecontainer 210, or back through valves 222 to the reagent enclosures 214.

FIG. 3 includes an illustration of a more detailed embodiment of thefluidic circuit. FIG. 3 diagrammatically illustrates a system employingan enclosure 614 that is a reagent reservoir, for example, for carryingout pH-based nucleic acid sequencing. Each electronic sensor of theapparatus generates an output signal. The fluid circuit permits multiplereagents to be delivered to the reaction chambers.

In FIG. 3, the system includes a fluidics circuit 602 connected to thereagent reservoirs 614, to a waste reservoir 620, and to a biosensor 634by fluid pathway 632 that connects fluidics node 630 to inlet 638 ofbiosensor 634 for fluidic communication. The prepared and mixed reagentsolution from reservoirs 614 can be driven to fluidic circuit 602 by avariety of methods including pressure, pumps, such as syringe pumps,gravity feed, and the like, and are selected by control of valves 650.Reagents from the fluidics circuit 602 can be driven to the wastecontainers 620 and 636. The control system 618 includes controllers forvalves 650 that generate signals for opening and closing via anelectrical connection 616.

The control system 618 also includes controllers for other components ofthe system, such as a wash solution valve 624 connected thereto by theelectrical connection 622, and the reference electrode 628. The controlsystem 618 can also include control and data acquisition functions forthe biosensor 634. In one mode of operation, the fluidic circuit 602delivers a sequence of selected reagents 1, 2, 3, 4, or 5 to thebiosensor 634 under programmed control of the control system 618, suchthat in between selected reagent flows, the fluidics circuit 602 isprimed and washed with a wash solution 626, and the biosensor 634 iswashed with the wash solution 626. Fluids entering the biosensor 634exit through the outlet 640 and are deposited in the waste container636. A similar setup may be used for optical sequencing systems, withphotodiodes or CCD cameras, for example.

In a particular example, the wash solution 626 can be a bufferedsuspension including the solid buffer particulate. The buffer suspension(wash solution) can be filtered using a filter 660 before entering thefluidics circuit 602 or sensor 634. In a further example, the bufferedsuspension can be applied to the reagent reservoirs 614 through filter662 to form the reagent solutions from reagent concentrate within thereagent reservoirs. Alternatively, the filter 660 and 662 can be thesame filter. In an example, the reagent concentrate is a liquidconcentrate. In another example, the reagent concentrate is a driedconcentrate, such as a lyophilized reagent (e.g., lyophilizednucleotides). Alternatively, the illustrated filters 660 and 662 can becombined. In another example, filters can be located downstream of thereagent reservoirs 614, such as between the reagent reservoirs 614 andthe valves 650.

FIG. 4 is a perspective view describing an exemplary reagent storageapparatus 1100. In an example, the reagent storage apparatus 100 caninclude an enclosure 1110. A container 1120 is disposed within theenclosure 1110. In an example, the enclosure 1110 can be a flexibleenclosure. A flexible enclosure, such as a sealable flexible bagenclosure, can be pressurized and depressurized by externally applyingpressure, such by applying pressurized gas to the external surfaces ofthe flexible enclosure. Alternatively, the enclosure can be rigid sothat externally applied gas pressure does not readily translate topressure of fluid within the enclosure 1110.

The reagent storage apparatus 1100 can also include a fluid port 1130coupled to a fitting 1160 attached to the enclosure 1110 to providefluid access to the interior of the enclosure 1110. The fluid port 1130can be coupled to the fitting 1160 to seal the enclosure 1110 from anexterior environment after insertion of the container 1120. Theenclosure 1110 can be, for example, thermosealed to itself and thefitting 1160, except where otherwise sealed by the fluid port 1130.

The container 1120 can include one or more arms 1140 and a flange 1150.The arms 1140 can position the container 1120 within the enclosure 1110,such as approximately centrally, to disperse a reagent within theenclosure 1100 evenly. The flange 1150 can be provided for convenientassembly of the container 1120. In an example, the arm 1140 is flexible.For example, the arm 1140 can be formed of wire or a polymeric material.Alternatively, the arm 1140 can be rigid. Alternatively, the arm 1140and the flange 1150 are not limited to those illustrated in FIG. 4 andcan include a structure that positions the container within apredetermined location or orientation within the enclosure 1110. Thecontainer 1120 can be directly or indirectly connected to the fluid port1130 or alternatively positioned a suitable distance away from the fluidport 1130 as show in FIG. 4. The sealed enclosure 1100 including theenclosure 1110 and container 1120 provides simplified storage andtransportation of a reagent within the container 1120.

FIG. 5 is a perspective view describing an example container 1200. Thecontainer 1200 can include a first portion 1210 and a second portion1220 coupled to the first portion 1210. Elements such as an optionalcompressible member and a reagent can be inserted within the container1200 prior to connecting the first portion 1210 to the second portion1220. In an example, the second portion 1220 can be a cap that slidesover or otherwise covers part of the first portion 1210 to form theinternal cavity. In another example, the second portion 1220 can be aninsert that slides into the first portion 1210. The second portion 1220can be connected to the first portion 1210 by any suitable attachmentmechanism including screwing the second portion 1220 onto the firstportion 1210 or vice versa, a locking mechanism, adhesive, or any othersuitable attachment mechanism.

In an example, the internal cavity defines a compressible volume. Thecompressible volume compresses in response to fluid pressure and doesnot dissipate or leave the internal cavity of the container 1200. Thecompressible volume can include a compressible gas volume or can be acompressible member, such as a resilient polymer or foam.

The container 1200 can define a passage 1230 providing fluidiccommunication between an internal cavity of the container 1200 and anexterior of the container 1200. In an example, one or more passages 1230can be defined through to the internal cavity. Such passages 1230 can bedrilled through the first or second portions of the container 1200. Inanother example, the second portion 1220 can include the passage 1230 orcan include a slot extending beyond a region at which the second portion1220 engages the first portion 1210, thus forming the passage 1230.

One or more arms 1240 can be coupled to the first portion 1210 toposition the container 1200 as desired within an enclosure. A flange1250 can be coupled to the second portion 1220 to assist with applyingthe second portion 1220 to the first portion 1210 or to position thecontainer 1200 away from a bottom of the enclosure.

FIG. 6 is a cross-sectional perspective view describing an examplecontainer 1300. The container 1300 defines an internal cavity 1320 anddefines a passage 1330 providing fluidic communication between theinternal cavity 1320 and the exterior of the container 1300. The passage1330 can be drilled through the container 1300. In another example, acap or insert can include a slot that extends beyond a region engagingthe container 1300 and that forms the passage 1330. The container 1300can include a first portion 1310 and a second portion 1350 coupled tothe first portion 1310 that allows elements such as a compressiblemember 1340 and reagent to be inserted into the internal cavity 1320 ofthe container 1300.

The internal cavity 1320 defines a compressible volume. The compressiblevolume is a volume that compresses in response to pressure to match thepressure, and can expand in response to depressuring, providing acounter force on fluid pressure. In an example, the compressible volumeincludes a compressible gas that compresses to match the pressure offluid entering the internal volume without dissipating or exiting theinternal cavity and in response to a depressurization of the fluidpushes the fluid out of the internal cavity 1320. Optionally, thecompressible volume can include a compressible member 1340. Thecompressible member 1340 is compressible under pressurization and upondepressurization, substantially returns to its previous form. Forexample, the compressible member 1340 can be a foam material. Inparticular, the compressible member 1340 can be a closed-cell foam ofelastomeric material. In an example, the compressible member can includepolyurethane foam.

In an example, the reagent can be disposed within the second portion1350. The reagent can be a lyophilized nucleotide or an analog thereof.In another example, the reagent is a solution absorbed on a porousmetal, ceramic, or polymeric sponge-like material or frit. Optionally,the reagent solution can be frozen. In an alternative example, thereagent can include a pH-adjusting reagent, such as an acid or base.

One or more arms 1360 can be coupled to the first portion 1310 toposition the container 1300 as desired within an enclosure. A flange1370 or other suitable appendage can be coupled to the second portion1350 to assist with engaging the second portion 1350 with the firstportion 1310 or to position the container 1300 within the enclosure.

FIG. 7 is an exploded schematic view describing an example container1400. The container 1400 can include a first portion 1410 and a secondportion 1420 (e.g., an insert) coupled to the first portion 1410,allowing elements such as an optional compressible member 1430 to beinserted within the container 1400. The second portion 1420 can besecured to the first portion 1410 by sliding or screwing into the firstportion 1410. One or more flexible arms 1440 can be coupled to the firstportion 1410 to position the container 1400 within an enclosure. Aflange 1450 can be coupled to the second portion 1420 to assist withengaging the second portion 1420 and the first portion 1410 or toposition the container 1400 within the enclosure.

In an example, the second portion 1420 can define one or more slots1460. The first portion 1410 and the second portion 1420 can engage soas to leave a portion of the one or more slots 1460 exposed, providingone or more passages between the internal cavity of the container 1400and the exterior of the container 1400.

FIG. 8 is a detailed perspective view describing an example container1500. The detailed view of the container 1500 can define a passage 1510providing fluidic communication between an internal cavity of thecontainer 1500 and an exterior of the container 1500. An end of thecontainer 1500 includes a fitting 1530 to receive an insert 1540. Theinsert 1540 includes a hole or slot not covered when the insert 1540 isapplied to the fitting, forming the passage 1510. Alternatively, theinsert 1540 can include a hole, notch, mesh, pores or any other suitablefeature for providing fluid communication to the fitting 1530. A flange1520 can be coupled to the container 1500 to allow control of the insert1540 as it is applied to the fitting 1530 and to position the containerwithin an enclosure.

FIG. 9 is a perspective view describing an exemplary reagent storageapparatus 1600. The reagent storage apparatus 1600 includes an enclosure1610. A container 1620 is disposed within the enclosure 1610. Theenclosure 1610 can be a flexible enclosure as described above. Forexample, the flexible enclosure can be a sealable flexible bag enclosurethat can be pressurized and depressurized externally via fluid pressureor gas pressure. Alternatively, the enclosure 1610 can be a rigidenclosure. The enclosure 1610 can sealably engage a seal structure 1670,such as a fitting, having a bore 1680, such as a central bore. Thecontainer 1620 can be coupled to an arm 1640, which can be coupled to afluid port 1630, and inserted through the bore 1680 of the fitting 1670.

The fluid port 1630 provides fluid access to the interior of theenclosure 1610 through the bore 1680. The fluid port 1630 can be coupledto the seal structure or fitting 1670 of the reagent storage apparatus1600 from an exterior environment after inserting the container 1620.The arm 1640 couples the container 1620 to the fluid port 1630 toposition the container 1620, for example, approximately centrally withinthe enclosure 1610 to disperse a reagent within the enclosure 1610evenly. The arm 1640, the container 1620, and the fluid port 1630 can bea single integrated piece. In an example fluid flows through the fluidport 1630 and through the bore 1680 of the fitting 1670 into theenclosure 1610, optionally along the arm 1640. The arm 1640 can positionthe container 1620 with or without a flange 1650.

FIG. 10 is a perspective view describing an example container 1700. Thecontainer 1700 can include a first portion 1710 and a second portion1720 coupled to the first portion 1710 allowing elements such as anoptional compressible member to be inserted within the container 1700. Afluid port 1730 is coupled to the container 1700 and provides fluidaccess to an enclosure into which the container 1700 is inserted. An arm1740 can coupled the first portion 1710 and the fluid port 1730 toposition the container 1700 within an enclosure.

In an example, the second portion 1720 is an insert to engage the firstportion 1710. In another example, the second portion 1720 forms a cap tocover an end of the first portion 1710. Fluid can flow through theopening 1770 of the port 1730 and to an opening 1760 along the arm 1740.The fluid port can include a gasket to facilitate sealing. A flange 1750can be coupled to the second portion 1720.

FIG. 11 is a cross-sectional perspective view describing an examplecontainer 1800. The container 1800 defines an internal cavity 1820 anddefines a passage 1830 providing fluidic communication between theinternal cavity 1820 and the exterior of the container 1800. Thecontainer 1800 can include a first portion 1810 and a second portion1850 coupled to the first portion 1810 defining a compressible volume.In an example, elements, such as a compressible member 1840 and reagent,can be inserted into the internal cavity 1820 of the container 1800.

In an example, the second portion 1850 is a cap to apply over an end ofthe first portion 1810. In another example, the second portion 1850 isan insert to apply to a fitting of the first portion 1810. In anexample, a passage 1830 is formed in the second portion 1850, forexample, as a hole or a slot.

The reagent can be disposed within the second portion 1850. The reagentcan be a lyophilized nucleotide or an analog thereof. In anotherexample, the reagent can be a nucleotide solution absorbed by a porousmetallic, ceramic or polymeric sponge or frit. In a further example, thereagent can be frozen. In an additional example, the reagent can includea pH-adjusting reagent, such as an acid or a base.

A fluid port 1860 is coupled to the container 1800 and provides fluidaccess to an enclosure in which the container 1800 is inserted. Forexample, fluid entering opening 1890 can pass through passages 1895 andinto an enclosure. An arm 1840 can be coupled to the first portion 1810and the fluid port 1860. A flange 1880 can be coupled to the secondportion 1850 to also position the container 1800 within the enclosure.

The reagent storage apparatus can be inserted into a case or cartridgehaving a cavity. In an example, pressure can be varied within the cavityto change the pressure of liquid within the flexible enclosure and thus,influence the pressure within the container. Alternatively, pressure canbe applied through the opening 1890 and internal to the enclosure. In anexample, one or more of the enclosures can be incorporated into thecase. The case can define one or more pressure chambers in whichpressure can be applied and relieved from the enclosures.

In a particular example illustrated in FIG. 12, a cartridge or case 1900includes a lid 1902 and a body 1904. The lid 1902 can receive the fluidports (1906, 1908, 1910, 1912, or 1914) of containers inserted intoflexible enclosures. The container can include different reagents. Forexample, each container can include a nucleotide or can include apH-adjusting reagent. The lid 1902 can also include a port 1916 forproviding pressurized gas or relieving the pressure, controlling thepressure outside of each of the enclosures and thereby controllingpressure within the enclosure. The walls of the base 1904 and the lid1902 can be configured to permit pressurizing a cavity within thecartridge 1900, for example, with pressurized gas or air. In an example,the cartridge 1900 can be labeled with a bar code or radio frequencyidentification (RFID) tag.

As illustrated in FIG. 12 and FIG. 13, the lid 1902 can include accessports 1918 and 1920 for applying gas or air through a scrubbercartridge. In particular, system can utilize external air, applying theexternal air through the port 1918 and receiving a cleaned gas or airthrough the port 1920. In particular, the scrubber cartridge can includeabsorbent materials for capturing carbon dioxide or water. Carbondioxide can be removed from air to prevent acidification of liquidcomponents when carbon dioxide diffuses into the enclosures or when theair is used in other parts of the system.

In a further example, the lid 1902 can also include alignment features1924 or 1926. Such alignment features can be used to align access to theports (1906, 1908, 1910, 1912, 1914, 1916, 1918, or 1920) with amanifold to limit damage to the manifold or provide for adequateengagement between the manifold and the case 1900.

As illustrated in FIG. 14, FIG. 15, and FIG. 16, the body 1904 candefine individual cavities 2126 into which each enclosure 2128 is placedand the nucleotide container 2130 is inserted. In an example, eachenclosure 2128 is disposed within individual cavities 2126 and eachcontainer 2130 is applied through the lid 1902, engaging the lid 1902 atthe fluid port of the container 2130. A fitting 2134 of the enclosures2128 can engage the lid 1902.

The lid 1902 can define a headspace that provides communication betweenthe pressurized gas input port 1916 and each of the cavities 2126.Alternatively, the cavity can be an open cavity absent individualizedcavities 2126 and provide a single cavity to which pressurized gas canbe applied to apply pressure to the enclosures 2128. As illustrated inFIG. 15, the body 1904 can include a chamber 2232 to receive a scrubbercartridge, for example, for removing carbon dioxide from the air.

In a top view, as illustrated in FIG. 16, the body 1904 includesindividualized cavities 2126. In addition, the body can include a sealstructure 2340 to isolate the scrubber cartridge input and output fromthe pressure of the rest of the body 1904. In addition, an internal seal2342 can be utilized to isolate the input pressure of air entering thescrubber cartridge from the output pressure of air leaving scrubbercartridge. Further, the body 1904 can include a seal structure 2344 toengage an opposing seal structure on the lid 1902 to provide an isolatedinterior space including the cavities that can be pressurized ordepressurized.

The containers can include nucleotide reagents or other reagents. Inparticular, individual containers within the cartridge system caninclude one of four nucleotides. The system can also include a containerwithin an enclosure that includes pH-adjusting reagents. In a particularexample, the cartridge includes containers and enclosures incorporatingeach of the four nucleotides (A, G, C, or T) and optionally, apH-adjusting reagent container. In an example, the reagents are in driedform. For example, lyophilized nucleotides can be stored within thecontainer. In another example, a reagent solution can be absorbed withina porous metallic, ceramic, or polymeric sponge-like material or frit.In a further example, the reagent solution can be frozen either within acontainer or within the porous sponge-like material into which thereagent solution is absorbed.

The enclosures described herein can be applied to prepare a reagentsolution. Assembly of the enclosure includes inserting a container intothe enclosure and sealing the container within the enclosure with afluid port. One or more enclosures can be further secured into a volumeof a case, where the case includes a gas port for providing external gaspressure to the secured enclosures. The enclosures can be inserted intoa case as a final assembly step or at a point just prior to mixing thatprovides flexibility of in the selection of reagents.

Alternatively, the enclosure can be secured to the lid prior toinserting the containers including reagent. The reagent containers canbe inserted through the lid and the fluid port of the containers canengage the lid. The lid can be secured to the base following securingthe enclosures to the lid or following inserting the containers throughthe lid into the enclosures.

The pressurization and depressurization of the fluid within theenclosures are controlled by increasing and decreasing the gas pressureof the volume of the case via the gas port.

A method for preparing a reagent solution includes filling an enclosure,such as any of the enclosures described herein including a container anda reagent, with a predetermined amount of fluid through a fluid port ofthe enclosure. The fluid within the enclosure is then pressurized suchthat fluid flows into the internal cavity of the container through apassage of the container. The fluid can be pressurized directly througha port. In another example, the fluid can be pressurized by applyingexternal pressure to the enclosure, for example, using gas or otherfluidic pressure. The pressurization compresses the compressible volumeor member within the internal cavity of the container while the fluidfills a portion of the volume of the internal cavity.

For example, the fluid flows into the internal cavity of the containerand compresses the compressible volume or member until the pressurewithin the internal cavity and exerted on the compressible volume ormember is approximately equal to a pressure within the enclosure andexternal to the container.

After reaching a predetermined pressure, the fluid within the enclosureis depressurized. The compressible volume or member decompresses so asto expand and eject the fluid and reagent from the internal cavity intothe enclosure outside of the container. The mixture of reagent and fluidejected from the passage creates eddy currents and turbulence within thebag enclosure sufficient to mix the reagent with fluid. Upondepressurization, a pressure within the internal cavity as imposed bythe compressible volume or member is greater than a pressure within theenclosure and external to the container. The fluid and the reagent ejectfrom the internal cavity through the passage until the pressure withinthe internal cavity is approximately equal to a pressure within theenclosure and external to the container to provide a well-mixed reagentsolution.

Pressurization can be performed by increasing a gas pressure external toa flexible enclosure. In one implementation, the enclosure can bedisposed within a case. Pressure within the enclosure can be controlledby increasing/decreasing a gas pressure within the case and external tothe enclosure. Proper mixing of the reagent and fluid can beaccomplished through repeated cycles of pressurization anddepressurization. After mixing is completed, the fluid and the reagentare released through the fluid port of the enclosure.

FIG. 17 and FIG. 18 illustrated an exemplary method for assembling areagent cartridge. For example, a fitting 702 can be secured to a bagenclosure form an enclosure 708. A plurality of enclosures 708 can becoupled to a lid 706 of a cartridge and inserted into a cartridge base704, as illustrated 714, when the cartridge lid 706 is secured to thecartridge base 704. A port gasket 710 can be secured to the cartridgelid to permit connection of the compressed air system to the cartridge.In a further example, a port gasket 712 can be secured to the cartridgelid 706 to permit access to a CO₂ scrubber.

Turning to FIG. 18, reagent containers can be formed by inserting anoptional compressible member into a first portion 716 of the reagentcontainer, as illustrated at 722. A second part 718 can be secured tothe first part 716 to form the reagent container 728. Optionally, thereagent is applied to the second part 718. Alternatively, the reagent isinserted into the first portion at 722.

A scrubber container 720 can be filled with scrubbing reagent to removeCO₂, for example, as illustrated at 726. A plurality of reagentscontainers 728 and the scrubber container 720 can be inserted into thereagent cartridge through the lid, as illustrated 730. The ends of thereagent cartridge containers 728 are supplied through the cartridge lidand into the interior of the reagent enclosures. The scrubber container720 is inserted through the lid and into an isolated compartment of thecartridge that permits air to flow in and out without influencing thepressure in the remaining cavity of the cartridge. Fluid port gaskets724 are secured over the reagent containers and optionally the scrubbercontainer to provide for fluid tight access to the reagent containers orscrubber container when secured within a manifold and instrument.

FIG. 19 provides an exploded schematic view of an example pinch valveregulator 3100. The valve 3100 includes a housing base 3110 and ahousing cover 3120 disposed above the base 3110. A diaphragm 3130 isdisposed between the housing base 3110 and the housing cover 3120. Apinch plate 3140 is disposed between the diaphragm 3130 and the base3110. In operation, the pinch plate 3140 moves relative to the housingbase 3110 to pinch a pinch tube 3150 against a pinch structure (asillustrated more clearly in FIGS. 20, 21 and 22) to restrict fluid flowthrough the pinch tube 3150. One or more gaskets 3160, 3170 can bedisposed between the housing base 3110 and housing cover 3120 to preventfluid leakage and to ensure smooth valve operation.

FIG. 20 provides a cross-sectional schematic view of an example pinchvalve regulator. A valve 3200 includes a housing base 3210 and a housingcover 3220 disposed over of the base 3210. The housing base 3210includes a lower cavity 3212 and a pinch structure 3214 protrudingwithin the lower cavity 3212. The housing base 3210 includes a gas inlet3230, providing external access to the lower cavity 3212. A base fluidinlet 3232 provides an external access path that is connected to one endof a pinch tube 3240 within the lower cavity 3212. The other end of apinch tube 3240 is connected to a base fluid outlet 3234. Accordingly,the pinch tube 3240 provides fluidic communication between the basefluid inlet 3232 and the base fluid outlet 3234. The pinch tube 3240extends between the pinch structure 3214 and a pinch point 3252 of apinch plate 3250.

In an example, the pinch structure 3214 includes a rectangular prismextending into the lower cavity 3212. As illustrated, the rectangularprism as a rounded top. In another example, the rectangular prism canhave a flat top. Alternatively, the prism can have a pointed structure,such as a triangular prism. In general, the pinch structure 3214 forms acounter structure to which the pinch point 3252 can secure and punch thepinch tube 3240.

The base fluid outlet 3234 is in turn connected to and provides fluidiccommunication with a cover fluid inlet 3236 between the upper cavity andthe lower cavity to provide a fluid path through the housing base 3210and the housing cover 3220. The cover fluid inlet 3236 is in fluidiccommunication with a cover fluid outlet 3238 via a fluid path 3270. Thecover fluid outlet 3238 provides external access to the fluid path 3270from the housing cover 3220. A diaphragm 3260 is disposed between thehousing base 3210 and the housing cover 3220 to fluidically separate thelower cavity 3212 from an upper cavity 3222 defined between the cover3220 and the diaphragm 3260.

The housing cover 3220 defines an upper cavity 3222 where the fluid path3270 is disposed. Optionally a gasket 3280 can define part of the lowercavity 3212 or part of the upper cavity 3222. The pinch plate 3250 canbe disposed within the cavity region defined by the housing cover 3220or the gasket 3280. The base fluid outlet 3234 and the cover fluid inlet3236 are in fluidic communication through the gasket 3280 and diaphragm3260. Alternatively, the base fluid outlet 3234 and the cover fluidinlet 3236 can be fluidically connected external to the housing base3210 or the housing cover 3220. The diaphragm 3260 provides separationbetween the lower cavity 3212 and the upper cavity 3222. A pinch plate3250 is disposed within the cavities 3212, 3222 defined within thehousing cover 3220 and the housing base 3210. The pinch plate 3250includes a pinch point 3252 that is disposed opposite the pinchstructure 3214. The pinch point 3252 is illustrated with a rounded tip.Alternatively, the pinch point 3252 can have a sharp tip. The pinchplate 3250 moves relative to the housing base 3210 to pinch the pinchtube 3240 to restrict fluid flow through the pinch tube 3240 based onfluid pressure within the fluid path 3270 and gas pressure within thelower cavity 3212.

The valves described herein operate to regulate fluid flow as a functionof gas pressure within the lower cavity. FIG. 20 illustrates a valvestructure prior to applying fluid into the valve 3200 and FIGS. 21-22illustrate an equilibrium state of a valve where fluid flows throughvalve 3300 at a flow rate based on the input gas pressure. Animplementation of the pinch valve in operation will be described belowwith reference to FIG. 20. FIG. 21 and FIG. 22.

Gas pressure is applied to a gas inlet 3230, 3330 of the valve topressurize the lower cavity 3212, 3312 at an input/reference gaspressure. The pressurized lower cavity applies an upward force againstthe pinch plate 3250, 3350 and diaphragm 3260, 3360 towards the housingcover 3220, 3320. Fluid is applied to the base fluid inlet 3332 andflows sequentially through pinch tube 3340, base fluid outlet 3334,cover fluid inlet 3336, fluid path 3370, cover fluid outlet 3338, andthen out of the valve. The fluid flowing through the housing cover 3320applies a downward force against the diaphragm 3360 and pinch plate 3350towards the housing base 3310. As the fluid pressure in the fluid path3370 increases relative to the gas pressure in the lower cavity 3312,the diaphragm 3360 moves toward the housing base 3310 and appliesdownward force against the pinch plate 3350. In particular, thediaphragm 3360 is to motivate the pinch point 3352 relative to the pinchstructure 3314 in response to a difference between a fluid pressure inthe upper cavity 3322 and a gas pressure in the lower cavity 3312. Forinstance, the diaphragm 3360 is to motivate the pinch point 3352 towardsthe pinch structure 3314 in response to an increase in the fluidpressure within the upper cavity 3322 relative to the gas pressure inthe lower cavity 3312 to restrict fluid flow in the pinch tube 3340.

As the pinch plate 3350 moves toward the housing base 3310, the pinchpoint 3352 applies a downward force onto pinch tube 3340 so as to pinchthe tube 3340 against the pinch structure 3314 and restrict fluid flowor cause a pressure drop across the punch tube 3340 and in the uppercavity 3322 until the input gas pressure counteracts the fluid pressurein the upper cavity 3322 to thereafter provide a constant fluid flowrate from the valve 3300. FIG. 22 illustrates a valve 3300 withdirectional arrows 3380 indicating the fluid flow path through the valve3300.

The pinch actuation force of the diaphragm driven pinch valve is suchthat the output fluid pressure is regulated by the input gas pressure.By setting the pressure in the lower gas cavity 3312 to a known value,fluid flow and pressure exiting the housing cover 3320 is controlled. Inthis manner, the valve self-regulates to reach equilibrium and canprovide a desired constant fluid flow. In summary, the output fluidpressure at the cover fluid outlet follows the input gas pressure at thegas inlet and can be independent of the fluid pressure at the base fluidinlet.

FIG. 23 and FIG. 24 include illustrations of an exemplary sequencingsystem that includes a manifold 806 to receive a cartridge 804. Themanifold may be driven up and down using an actuator 808. Optionally, awaste vessel 802 can be positioned within the instrument and fluidicallyconnected to the cartridge 804, for example, as illustrated in theschematic of FIG. 2.

As illustrated in FIG. 24, a plurality of fluid ports 810 can beconnected to tubing and a fluidic circuit, such as the fluidicsschematic illustrated in FIG. 2. The cartridge 804 can optionallyinclude an RFID tag readable by the instrument. Connection of themanifold 806 to the cartridge 804 can be prevented or permitted based onthe reading of the RFID tag.

FIG. 25 diagrammatically illustrates another embodiment of the fluidicscircuit of the invention which accommodates five input reagents in aplanar circuit structure. FIG. 25 is a top view of a transparent body orhousing 4300 containing fluidic circuit 4302. A housing can beconstructed from a variety of materials, including metals, glass,ceramics, plastics, or the like. Transparent materials includepolycarbonate, polymethyl methacrylate, and the like. Inlets (or inputports) are connected by a passage to their respective connector slots(e.g. 4370) located on the bottom side of housing from which reagentsenter fluidic circuit 4302. Inlets are in fluid communication withpassages (e.g., 4353) which, in turn, are connected to curvilinearpassages. Each curvilinear passage consists of two legs, identified forcurvilinear passage at a “T” junction 4356. One leg is an inner legwhich connects its respective inlet to node (or multi-use central port)4301 and the other leg is an outer leg which connects its respectiveinlet to waste passage (or ring) 4340. As mentioned above, thecross-sectional areas and lengths of the inner and outer legs of thecurvilinear passages may be selected to achieve the desired balance offlows at the “T” junctions and at node 4301. Through passage, wastepassage (or channel) 4340 is in fluid communication with waste port 4345which connects to a waste reservoir (not shown) by connector slot on thebottom side of body. Node 4301 is in fluid communication with port 4363by passage 4361 which in this embodiment is external to body 4300 and isillustrated by a dashed line. In other embodiments, passage 4361 may beformed in body so that connector slots for node 4301 and port 4363 arenot required. Port is connected by passage 4363 to wash solution inlet,where a “T” junction is formed, and to connector slot which, in turn,provides a conduit to a flow cell, reaction chamber, or the like. FIG.25 illustrates a mode of using the fluidics circuit to distribute fluidsto a flow cell. The modes of operation are implemented by valves 4350associated with each of the input reagents and with the wash solution.In a first mode of operation (selected reagent valve open, all otherreagent valves closed, wash solution valve closed) a selected reagent isdelivered to a flow cell; in a second mode of operation (selectedreagent valve open, all other reagent valves closed, wash solution valveopen) the fluidic circuit is primed to deliver a selected reagent; andin a third mode of operation (all reagent valves closed wash solutionvalve open) (not shown), all passages in the fluidics circuit arewashed. As mentioned above, associated with each inlet is a valve 4350which can be opened to allow fluid to enter fluidic circuit 4302 throughits respective inlet (as shown for valve (4352)), or closed to preventfluid from entering circuit 4302 (as shown with all valves, except for4352). In each case, when an inlet's valve is open and the others areclosed (including the wash solution valve) as shown for inlet 4370 inthe FIG. 25, fluid flows through passage 4354 to “T” junction 4356 whereit is split into two flows, one of which is directed to waste passage4340 and then the waste port 4345, and another of which is directed tonode 4301. From node 4301 this second flow again splits into multipleflows, one of which exits node 4301 through passage 4361 and then topassage 4363 and to a flow cell and the other flows to each of thepassages connecting node 4301 to the other inlets, and then to wastepassage 4340 and waste port 4345. The latter flows pass the other inletscarrying any material diffusing or leaking therefrom and directing it towaste port 4345. A sequence of different reagents may be directed to aflow cell by opening the valve of a selected reagent and simultaneouslyclosing the valves of all of the non-selected reagents and the washsolution. In one embodiment, such sequence may be implemented by asequence of operating modes of the fluidics circuit such as: wash, primereagent x1, deliver reagent x1, wash, prime reagent x2, deliver reagentx2, wash, and so on. For the reagent priming mode, as in the reagentdelivery mode, all reagent inlet valves are closed, except for the valvecorresponding to the selected reagent unlike the reagent delivery mode,however, the wash solution valve is open and the relative pressure ofthe selected reagent flow and the wash solution flow is selected so thatwash solution flows through passage 4361 and into node 4301 where itthen exits through all the passages leading to waste passage 4340,except for the passage leading to the selected reagent inlet.

As illustrated in FIG. 26, a method 900 for preparing a reagent solutionwithin a cartridge includes inserting the cartridge into an instrument,as illustrated 902. For example, the cartridge can be inserted under amanifold, similar to the embodiments illustrated in FIG. 23 or FIG. 24,or the instrument illustrated in FIG. 1.

Optionally, the system can check the cartridge alignment features, asillustrated 904. For example, the cartridge can include structures thatindicate proper positioning of the cartridge within the instrument. Asfurther illustrated at 906, that the cartridge can optionally include aradio frequency identifier tag (RFID tag) that can be read by theinstrument. Based on the test of the cartridge alignment features or thereading of the RFID tag of the cartridge, the instrument can selectivelyengage the cartridge using a manifold.

For example, the instrument can connect a plurality of fluid portssecured to the manifold to the cartridge, providing fluidic connectivitywith the rest of the instrument, as illustrated 908. The instrument canfurther connect a gas system, such as a compressed air system, to thecartridge, as illustrated 910. In an example, the connections to the gassystem are incorporated within the manifold that connects the fluidports to the reagent cartridges. In particular, the system can beconnected to the scrubber inlet and outlet and optionally to the cavitydefining space between the reagent enclosures.

As illustrated at 912, the cartridge cavity can be pressurized. Theenclosures or reagent enclosures can evacuate as a result of thepressurizing of the cartridge cavity. Following evacuation of theenclosures, the cartridge cavity can further be depressurized.

A solution (e.g., a buffered solution) can be applied through the fluidports, as illustrated at 914. In particular, the solution can beuniversally applied to each of the reagent enclosures. Followingapplication of the solution, the pressure within the cartridge cavityand can then be cycled as illustrated at 916. Such cycling can causemixing of reagents within the enclosures, for example utilizing themechanisms described above in relation to FIGS. 4-16.

FIG. 27 illustrates an exemplary method 920 for performing measurementsof an analyte, followed by cleaning the system. For example, asillustrated at 922, pressure can be applied to a cartridge cavity,pressurizing reagent enclosures.

As illustrated at 924, reagents can selectively flow from the reagentenclosures by opening valves connecting individual reagent enclosures tothe fluidic circuit. In particular, reagent can flow from selectindividual reagent enclosures in sequential orders, optionally separatedby the flow of the wash solution from a separate container.

While the pressure of the cartridge cavity can provide a driving forcefor reagents from the reagent enclosures and individual reagent can beselected by selectively opening a valve associated with a reagentenclosure, the flow rate can be controlled, as illustrated 926,downstream of the fluidics circuit. In particular, flow can becontrolled downstream of the fluidic circuit by controlling flow to thewaste containers, for example, utilizing pinch flow regulators.

The system can then be clean by applying pressurized air through thescrubber of the cartridge, as illustrated 928, and flowing thepressurized scrubbed air through the fluidic circuit 930. Thepressurized air can drive fluid from the fluidic circuit to the wastecontainers, for example through the sensor device and pinch flowregulators. In a further example, the pressurized air can drive reagentfluids backwards through their associated valves and into the reagentenclosures within the cartridge. In such a case, the cartridge cavitycan be depressurized, allowing the pressurized air to drive the reagentfluids backwards into the reagent enclosures.

As illustrated in FIG. 28, a method 940 for preparing a reagentcartridge includes attaching a plurality of reagent enclosures to acartridge lid, as illustrated 942. An exemplary method is provided shownor illustrated in more detail in schematic of FIG. 17 and FIG. 18. Thecartridge lid can be secured to a cartridge base, as illustrated 944,and a plurality of reagent containers can be inserted into the pluralityof enclosures through the cartridge lid, as illustrated 946.

The system or instrument can be integrated into a process flow forsequencing. For example, the system can be utilized with the One Touch2® or Ion Chef® systems, which perform template preparation. Theinstrument can be utilized for sequencing following the templatepreparation. The instrument is configurable to perform an initialanalysis or can outsource the initial analysis and interpretation to acloud or an external server.

The instrument can be configurable. For example, the system can includeone or more central processing units, configurable amounts of RAM,upgradable graphics processing units and exchangeable storage includingfrom 1 to 12 TB. The instrument is configured to receive and performsequencing using different sequencing chips. Further, the system can beupgradable to access external servers for analysis and interpretation ofthe data received from the sequencing chip.

Multiple chips supported by the instrument can also support multipleassays, allowing for a different number of reads, read lengths, baseoutputs and applications. As such, the system is versatile and useful ina variety of fields of research.

Such a system is provided for desirable sequencing runs including outputfrom a P1 proton chip including 19.6 Gb or a high accuracy run including15.4 Gb.

In a first aspect, a method of preparing reagents includes inserting acartridge into an instrument. The cartridge includes a plurality ofreagent enclosures disposed in a cavity of the cartridge and exposing aport to an exterior of the cartridge. Each reagent enclosure includes areagent container including a reagent and an internal cavity defining acompressible volume, an opening defined through the reagent container tothe internal cavity. The method further includes connecting a pluralityof fluid ports to the openings of the plurality of reagent enclosures;applying a solution through the fluid ports to at least partially fillthe plurality of reagent enclosures; and cycling a pressure of thecavity, whereby for each of the reagent enclosures, during increasingpressure, the solution enters the internal cavity of the reagentcontainer, combines with the reagent, and compresses the compressiblevolume, and during decreasing pressure, the compressible volumedecreases and the reagent is ejected through the opening.

In an example of the first aspect, the method further includespressurizing the cavity prior to applying the solution to remove gasfrom the plurality of enclosures.

In another example of the first aspect and the above examples, thereagent container further includes a compressible member disposed in thecompressible volume.

In a further example of the first aspect and the above examples, thereagent includes a nucleotide.

In an additional example of the first aspect and the above examples, themethod further includes sensing a position of the cartridge prior toconnecting the plurality of fluid ports.

In another example of the first aspect and the above examples, themethod further includes reading an identification tag of the cartridgewith the instrument. For example, the method can further includeconnecting the plurality of fluid ports based on the reading.

In a further example of the first aspect and the above examples, thecartridge further includes a scrubber, the method further comprisingconnecting a gas system to the scrubber. For example, the scrubber isCO2 scrubber. In another example, cycling the pressure includes applyinggas through the scrubber and into the cavity.

In a second aspect, a method of detecting an analyte with an instrumentincludes applying pressure to a cartridge coupled to an instrument. Thecartridge includes a plurality of reagent enclosures disposed in acavity of the cartridge and exposing a port to an exterior of thecartridge. Each reagent enclosure includes a reagent container includinga reagent and an internal cavity defining a compressible volume, anopening defined through the reagent container to the internal cavity.The method further includes selectively flowing a reagent from a reagentenclosure of the plurality of reagent cartridges from the cartridge,through a fluidic circuit, a sensor, and a pinch flow regulator to awaste container; and controlling flow to the waste container using thepinch flow controller.

In an example of the second aspect, applying pressure to the cartridgeincludes flowing gas through a scrubber in the cartridge and into thecavity of the cartridge.

In another example of the second aspect and the above examples, thesensor includes a ion sensitive field effect transistor.

In a further example of the second aspect and the above examples,applying pressure includes compressing air with a compressor of theinstrument.

In a third aspect, a method of cleaning an instrument includes applyingpressurized air through a scrubber of a cartridge; and flowing thescrubbed pressurized air through a fluidics circuit, portions of thescrubbed pressurized air pushing a fluid through the fluidics circuittoward reagent enclosures of the cartridge and a portion through asensor of the instrument through a pinch flow regulator to a wastecontainer.

In a fourth aspect, a method for preparing a reagent cartridge includesattaching a plurality of enclosures to a cartridge lid; securing thecartridge lid to a cartridge base, the cartridge lid defining aplurality of openings, an opening of the cartridge lid providing accessto an interior of an enclosure of the plurality of enclosures; andinserting a plurality of reagent containers into the plurality ofenclosures, a reagent container of the plurality of reagent containersextending through the opening to the interior of the enclosure, thereagent container including a reagent and an internal cavity defining acompressible volume, an access defined through the reagent container tothe internal cavity.

In an example of the fourth aspect, the reagent includes a nucleotide.

In another example of the fourth aspect and the above examples, thenucleotide is disposed on a porous material within the internal cavityof the reagent container.

In a further example of the fourth aspect and the above examples, themethod further includes inserting a scrubber into the cartridge prior tosecuring the cartridge lid.

In a fifth aspect, a system includes a cartridge manifold to connect toa cartridge and provide fluidic communication between a plurality ofreagent enclosures and a fluidics circuit and to provide fluidiccommunication between a compressed gas system, a cartridge cavity and ascrubber of the cartridge; the fluidics circuit; a sequencing device influidic communication with the fluidics circuit; a pinch flow regulatorin fluidic communication with the sequencing device; and a wastecontainer in fluidic communication with the pinch flow regulator.

Note that not all of the activities described above in the generaldescription or the examples are required, that a portion of a specificactivity may not be required, and that one or more further activitiesmay be performed in addition to those described. Still further, theorder in which activities are listed are not necessarily the order inwhich they are performed.

In the foregoing specification, the concepts have been described withreference to specific embodiments. However, one of ordinary skill in theart appreciates that various modifications and changes can be madewithout departing from the scope of the invention as set forth in theclaims below. Accordingly, the specification and figures are to beregarded in an illustrative rather than a restrictive sense, and allsuch modifications are intended to be included within the scope ofinvention.

As used herein, the terms “comprises,” “comprising,” “includes,”“including,” “has,” “having” or any other variation thereof, areintended to cover a non-exclusive inclusion. For example, a process,method, article, or apparatus that comprises a list of features is notnecessarily limited only to those features but may include otherfeatures not expressly listed or inherent to such process, method,article, or apparatus. Further, unless expressly stated to the contrary,“or” refers to an inclusive-or and not to an exclusive-or. For example,a condition A or B is satisfied by any one of the following: A is true(or present) and B is false (or not present), A is false (or notpresent) and B is true (or present), and both A and B are true (orpresent).

Also, the use of “a” or “an” are employed to describe elements andcomponents described herein. This is done merely for convenience and togive a general sense of the scope of the invention. This descriptionshould be read to include one or at least one and the singular alsoincludes the plural unless it is obvious that it is meant otherwise.

Benefits, other advantages, and solutions to problems have beendescribed above with regard to specific embodiments. However, thebenefits, advantages, solutions to problems, and any feature(s) that maycause any benefit, advantage, or solution to occur or become morepronounced are not to be construed as a critical, required, or essentialfeature of any or all the claims.

After reading the specification, skilled artisans will appreciate thatcertain features are, for clarity, described herein in the context ofseparate embodiments, may also be provided in combination in a singleembodiment. Conversely, various features that are, for brevity,described in the context of a single embodiment, may also be providedseparately or in any subcombination. Further, references to valuesstated in ranges include each and every value within that range.

What is claimed is:
 1. A method of preparing reagents, the methodcomprising: inserting a cartridge into an instrument, the cartridgeincluding a plurality of flexible reagent enclosures disposed in acavity of the cartridge and each reagent enclosure exposing a port to anexterior of the cartridge, each reagent enclosure including a reagentcontainer, the reagent container including an internal cavity defining acompressible volume, an opening defined through the reagent container tothe internal cavity, and a reagent contained in the internal cavity;connecting a plurality of fluid ports of the instrument to the reagentenclosures, a fluid port of the plurality of fluid ports connecting tothe port of one of the reagent enclosures; connecting a buffer solutioncontainer including buffer solution to the instrument; applying thebuffer solution through the fluid ports of the instrument to at leastpartially fill the plurality of reagent enclosures; and cycling apressure of the cavity by applying gas through a gas port of thecartridge, whereby for each of the reagent enclosures, during increasingpressure, the solution enters the internal cavity of the reagentcontainer, combines with the reagent, and compresses the compressiblevolume, and during decreasing pressure, the compressible volumedecreases and the reagent is ejected through the opening of the reagentcontainer to form a reagent solution.
 2. The method of claim 1, furthercomprising pressurizing the cavity prior to applying the solution toremove gas from the plurality of enclosures.
 3. The method of claim 1,wherein the reagent container further includes a compressible memberdisposed in the compressible volume.
 4. The method of claim 1, whereinthe reagent includes a nucleotide.
 5. The method of claim 1, furthercomprising sensing a position of the cartridge in the instrument priorto connecting the plurality of fluid ports.
 6. The method of claim 5,wherein sensing the position of the cartridge includes sensing alignmentfeatures on the cartridge.
 7. The method of claim 6, wherein thealignment features include structures that indicate positioning of thecartridge within the instrument.
 8. The method of claim 1, furthercomprising reading an identification tag of the cartridge with theinstrument after inserting the cartridge into the instrument.
 9. Themethod of claim 8, further comprising connecting the plurality of fluidports of the instrument based on the reading.
 10. The method of claim 1,wherein the cartridge further includes a scrubber in the cartridge, themethod further comprising connecting a gas system to the scrubber. 11.The method of claim 10, wherein the scrubber is CO2 scrubber.
 12. Themethod of claim 10, wherein cycling the pressure includes applying gasthrough the scrubber and into the cavity.
 13. The method of claim 1,further comprising: applying pressure to the cavity of the cartridgecoupled to the instrument; selectively flowing the reagent solution froma reagent enclosure of the plurality of reagent enclosures from thecartridge, through a fluidic circuit, a sensor, and a pinch flowregulator of the instrument to a waste container; and controlling flowto the waste container using the pinch flow regulator.
 14. The method ofclaim 13, wherein applying pressure to the cartridge includes flowinggas through a scrubber in the cartridge and into the cavity of thecartridge.
 15. The method of claim 13, wherein the sensor includes anion sensitive field effect transistor.
 16. The method of claim 13,wherein applying pressure includes compressing air with a compressor ofthe instrument.
 17. The method of claim 13, further comprising: applyingpressurized air through a scrubber of the cartridge; and flowing thescrubbed pressurized air through a fluidics circuit of the instrument, aportion of the scrubbed pressurized air pushing a fluid through thefluidics circuit toward reagent enclosures of the cartridge and aportion of the scrubbed pressurized air pushing a fluid through thesensor of the instrument through the pinch flow regulator to the wastecontainer.
 18. The method of claim 1, wherein the instrument includes amanifold to receive the cartridge.
 19. The method of claim 18, whereinthe instrument includes an actuator to drive the manifold to connectwith the cartridge.
 20. The method of claim 19, wherein the manifold isdriven to connect with the cartridge at least in part in response to areading of an identification tag associated with the cartridge.